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Chandipura virus (CHPV, a member of the Rhabdoviridae family) is an emerging pathogen that causes rapidly progressing influenza-like illness and acute encephalitis often leading to coma and death of the human host. Given several CHPV outbreaks in Indian sub-continent, recurring sporadic cases, neurological manifestation, and high mortality rate of this infection, CHPV is gaining global attention. The 'dark proteome' includes the whole proteome with special emphasis on intrinsically disordered proteins (IDP) and IDP regions (IDPR), which are proteins or protein regions that lack unique (or ordered) three-dimensional structures within the cellular milieu. These proteins/regions, however, play a number of vital roles in various biological processes, such as cell cycle regulation, control of signaling pathways, etc. and, therefore, are implicated in many human diseases. IDPs and IPPRs are also abundantly found in many viral proteins enabling their multifunctional roles in the viral life cycles and their capability to highjack various host systems. The unknown abundance of IDP and IDPR in CHPV, therefore, prompted us to analyze the dark proteome of this virus. Our analysis revealed a varying degree of disorder in all five CHPV proteins, with the maximum level of intrinsic disorder propensity being found in Phosphoprotein (P). We have also shown the flexibility of P protein using extensive molecular dynamics simulations up to 500 ns (ns). Furthermore, our analysis also showed the abundant presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs) in CHPV proteins. The identification of IDPs/IDPRs in CHPV proteins suggests that their disordered regions may function as potential interacting domains and may also serve as novel targets for disorder-based drug designs. [ABSTRACT FROM AUTHOR]

TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi’s sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection. [ABSTRACT FROM AUTHOR]

Argonaute-2 protein (Ago2), a major component of RNA-induced silencing complex (RISC), has been viewed as a cytoplasmic protein. In this study, we demonstrated by immunofluorescence confocal microscopy that Ago2 is distributed mainly as a nuclear protein in primary human foreskin keratinocytes in monolayer cultures and their derived organotypic (raft) cultures, although it exhibits only a minimal level of nuclear distribution in continuous cell lines such as HeLa and HaCaT cells. Oncogenic human papillomavirus type 16 (HPV16) or type 18 (HPV18) infection of the keratinocytes does not affect the nuclear Ago2 distribution. Examination of human tissues reveals that Ago2 exhibits primarily as a nuclear protein in skin, normal cervix, and cervical cancer tissues, but not in larynx. Together, our data provide the first convincing evidence that the subcellular distribution of Ago2 occurs in a cell type- and tissue context-dependent manner and may correlate with its various functions in regulation of gene expression. [ABSTRACT FROM AUTHOR]

An accurate method using image sharpness to determine the best focusing is proposed for ultra-high-voltage electron microscopy. This method maximizes image sharpness for adjusting the focus. Five images with different defocus values are used to calculate the image sharpness. To obtain the best focus value that produces greatest image sharpness, fitting the quasi-Gaussian function to five image sharpness is a suitable alternative. This method, which maximizes image sharpness, gives better accuracy than the wobbler method for the ultra-high-voltage electron microscope. The focusing area can be selected without moving the field of view, because the focusing area can be selected at almost any area in the image. [ABSTRACT FROM PUBLISHER]

Hepatitis C virus (HCV) entry into permissive cells is a com- plex process that involves interactions with at least four co-fac- tors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pre- treatment with an acidic buffer or with ECL2 enhanced infec- tion through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-spe- cific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate con- formations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection. [ABSTRACT FROM AUTHOR]

Abstract: The diagram of axis intercept (Nishi and Takaoka ) is extended to consider the energy dispersion characteristic. The operating condition of core-lenses can be optimized by taking into account not only the aperture aberration but also the chromatic aberration. The diagram is applied to a typical core-lens system configured with three point charges of different polarities and is plotted as a function of the electron energy in addition to the starting angle. Near the stigmatic condition, the diagram fits the quadratic dependence on both the energy dispersion and the beam tilt angle. Then, the coefficients of aperture aberration, energy dispersion and chromatic aberration are obtained. [Copyright &y& Elsevier]

Abstract. Noncontact imaging was successfully performed with the quartz resonator in air by using the FM detection method. A constant frequency shift mode was used. The frequency shill was about -30 mhz, which was induced by the attractive force gradient. A noncontact image of the Si(111) atomic step was obtained with vertical and lateral resolutions of 0.8 Angstrom and 60 Angstrom. [ABSTRACT FROM AUTHOR]

Noncontact imaging was successfully performed with the quartz resonator in air by using the FM detection method. A constant frequency shift mode was used. The frequency shift was about -30 mHz, which was induced by the attractive force gradient. A noncontact image of the Si(111) atomic step was obtained with vertical and lateral resolutions of 0.8 Å and 60 Å. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is an RNA-binding posttranscriptional regulator. We recently applied an affinity-purified anti-ORF57 antibody to conduct ORF57 cross-linking immunoprecipitation (CLIP) in combination with RNA-sequencing (CLIP-seq) and analyzed the genome-wide host RNA transcripts in association with ORF57 in BCBL-1 cells with lytic KSHV infection. Mapping of the CLIP RNA reads to the human genome (GRCh37) revealed that most of the ORF57- associated RNA reads were from rRNAs. The remaining RNA reads mapped to several classes of host noncoding and protein-coding mRNAs. We found that ORF57 binds and regulates expression of a subset of host long noncoding RNAs (lncRNAs), including LINC00324, LINC00355, and LINC00839, which are involved in cell growth. ORF57 binds small nucleolar RNAs (snoRNAs) responsible for 18S and 28S rRNA modifications but does not interact with fibrillarin or NOP58. We validated ORF57 interactions with 67 snoRNAs by ORF57 RNA immunoprecipitation (RIP)-snoRNA array assays. Most of the identified ORF57 rRNA binding sites (BS) overlap the sites binding snoRNAs. We confirmed ORF57-snoRA71B RNA interaction in BCBL-1 cells by ORF57 RIP and Northern blot analyses using a 32P-labeled oligonucleotide probe from the 18S rRNA region complementary to snoRA71B. Using RNA oligonucleotides from the rRNA regions that ORF57 binds for oligonucleotide pulldown-Western blot assays, we selectively verified ORF57 interactions with 5.8S and 18S rRNAs. Polysome profiling revealed that ORF57 associates with both monosomes and polysomes and that its association with polysomes increases PABPC1 binding to polysomes but prevents Ago2 association with polysomes. Our data indicate a functional correlation with ORF57 binding and suppression of Ago2 activities for ORF57 promotion of gene expression. [ABSTRACT FROM AUTHOR]

Summary: Electron tomography methods using the conventional transmission electron microscope have been widely used to investigate the three‐dimensional distribution patterns of various cellular structures including microtubules in neurites. Because the penetrating power of electrons depends on the section thickness and accelerating voltage, conventional TEM, having acceleration voltages up to 200 kV, is limited to sample thicknesses of 0.2 µm or less. In this paper, we show that the ultra‐high voltage electron microscope (UHVEM), employing acceleration voltages of higher than 1000 kV (1 MV), allowed distinct reconstruction of the three‐dimensional array of microtubules in a 0.7‐µm‐thick neurite section. The detailed structure of microtubules was more clearly reconstructed from a 0.7‐µm‐thick section at an accelerating voltage of 1 MV compared with a 1.0 µm section at 2 MV. Furthermore, the entire distribution of each microtubule in a neurite could be reconstructed from serial‐section UHVEM tomography. Application of optimised UHVEM tomography will provide new insights, bridging the gap between the structure and function of widely‐distributed cellular organelles such as microtubules for neurite outgrowth. Lay Description: An optimal 3D visualisation of microtubule cytoskeleton using ultra‐high voltage electron microscopy tomography The ultra‐high voltage electron microscope (UHVEM) is able to visualise a micrometre‐thick specimen at nanoscale spatial resolution because of the high‐energy electron beam penetrating such a specimen. In this study, we determined the optimal conditions necessary for microtubule cytoskeleton imaging within 0.7‐µm‐thick section using a combination with UHVEM and electron tomography method. Our approach provides excellent 3D information about the complex arrangement of the individual microtubule filaments that make up the microtubule network. [ABSTRACT FROM AUTHOR]

Connivance of cellular factors during virus‐host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin‐null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c‐src. However, the villin RRI mutant, lacking actin‐severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome‐CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell‐type dependent phosphorylation in regulating membrane fusion. [ABSTRACT FROM AUTHOR]

Abstract Adrenomedullin is a peptide hormone with multifunctional biological properties. Its most characteristic effects are the regulation of circulation and the control of fluid and electrolyte homeostasis through peripheral and central nervous system actions. Although adrenomedullin is a vasodilator of cerebral vasculature, and it may be implicated in the pathomechanism of cerebrovascular diseases, the source of adrenomedullin in the cerebral circulation has not been investigated thus far. We measured the secretion of adrenomedullin by radioimmunoassay and detected adrenomedullin mRNA expression by Northern blot analysis in primary cultures of rat cerebral endothelial cells (RCECs), pericytes and astrocytes. We also investigated the expression of specific adrenomedullin receptor components by reverse transcriptase-polymerase chain reaction and intracellular cAMP concentrations in RCECs and pericytes. RCECs had approximately one magnitude higher adrenomedullin production (135 ± 13 fmol/105 cells per 12 h; mean ± SD, n = 10) compared to that previously reported for other cell types. RCECs secreted adrenomedullin mostly at their luminal cell membrane. Adrenomedullin production was not increased by thrombin, lipopolysaccharide or cytokines, which are known inducers of adrenomedullin release in peripheral endothelial cells, although it was stimulated by astrocyte-derived factors. Pericytes had moderate, while astrocytes had very low basal adrenomedullin secretion. In vivo experiments showed that adrenomedullin plasma concentration in the jugular vein of rats was approximately 50% higher than that in the carotid artery or in the vena cava. Both RCECs and pericytes, which are potential targets of adrenomedullin in cerebral microcirculation, expressed adrenomedullin receptor components, and exhibited a dose-dependent increase in intracellular cAMP concentrations after exogenous adrenomedullin administration. Antisense oligonucleotide treatment... [ABSTRACT FROM AUTHOR]

Neuroblastoma arises from sympathoadrenal progenitors of the neural crest and expression of the neurotrophin receptor TrkB and its ligand, brain-derived neurotrophic factor (BDNF), is correlated with poor prognosis. Although activated TrkB signaling promotes a more aggressive phenotype in established neuroblastoma cell lines, whether TrkB signaling is sufficient to transform neural crest-derived cells has not been investigated. To address the role of TrkB signaling in malignant transformation, we removed two immunoglobulin-like domains from the extracellular domain of the full-length rat TrkB receptor to create a ΔIgTrkB that is constitutively active. In the pheochromocytoma-derived cell line PC12, ΔIgTrkB promotes differentiation by stimulating process outgrowth; however, in the rat neural crest-derived cell line NCM-1, ΔIgTrkB signaling produces a markedly transformed phenotype characterized by increased proliferation, anchorage-independent cell growth, anoikis resistance and matrix invasion. Furthermore, expression of ΔIgTrkB leads to the upregulation of many transcripts encoding cancer-associated genes including cyclind1, twist1 and hgf, as well as downregulation of tumor suppressors such as pten and rb1. In addition, ΔIgTrkB NCM-1 cells show a 21-fold increase in mRNA for MYCN, the most common genetic marker for a poor prognosis in neuroblastoma. When injected into NOD SCID mice, control GFP NCM-1 cells fail to grow whereas ΔIgTrkB NCM-1 cells form rapidly growing and invasive tumors necessitating euthanasia of all mice by 15 days post injection. In summary, these results indicate that activated TrkB signaling is sufficient to promote the formation of a highly malignant phenotype in neural crest-derived cells. [ABSTRACT FROM AUTHOR]

We have analysed the formation of streak artefacts in the reconstruction based on the filtered back projection algorithm in electron tomography (ET) and accordingly applied an adaptive interpolation technique to artefact reduction. In the adaptive interpolation to recover the missing information, the edge positions in a projection curve were tracked to reduce the interpolation error. A simulation was used to demonstrate the effectiveness of the artefact reduction. Furthermore, image reconstruction of integrated circuit specimens in the ET experiments with the ultra-high voltage electron microscope show that the strong streak artefacts can be reduced effectively by our artefact reduction technique. [ABSTRACT FROM AUTHOR]

Precision spectroscopy of the Muonium Lamb shift and fine structure requires a robust source of 2S Muonium. To date, the beam-foil technique is the only demonstrated method for creating such a beam in vacuum. Previous experiments using this technique were statistics limited, and new measurements would benefit tremendously from the efficient 2S production at a low energy muon ( < 20 keV) facility. Such a source of abundant low energy μ + has only become available in recent years, e.g. at the Low-Energy Muon beamline at the Paul Scherrer Institute. Using this source, we report on the successful creation of an intense, directed beam of metastable Muonium. We find that even though the theoretical Muonium fraction is maximal in the low energy range of 2–5 keV, scattering by the foil and transport characteristics of the beamline favor slightly higher μ + energies of 7–10 keV. We estimate that an event detection rate of a few events per second for a future Lamb shift measurement is feasible, enabling an increase in precision by two orders of magnitude over previous determinations. [ABSTRACT FROM AUTHOR]

The article reports on the multiple cases of Seoul virus infection (SVI) in a household with infected pet rats in Tennessee from December 2016 to April 2017. Topics mentioned include the symptoms of SVI including high fever and anorexia, the techniques for reducing SVI risk including avoiding unprotected contact with rodent urine, and the importance of adherence to recommendations for euthanasia.